Detection of nucleic acids has an important role in diagnosis of a variety of diseases. The conventional molecular techniques for detection of nucleic acids are usually time consuming and require laborious preparation of samples, including nucleic acid extraction. The use of those harsh procedures results in destruction of cellular morphology, which is a great disadvantage in cases when cellular localization of specific sequences is of particular interest.
Chicken anemia virus (CAV), formerly called chicken anemia agent (CAA), was first isolated by Yuasa et al..sup.1 and its viral particles were found to contain circular single-stranded DNA.sup.2,3. CAV causes severe aplastic anemia in chickens.sup.1,4,5, depletion of lymphoid organs, subcutaneous and intramuscular hemorrhages, and destruction of erythroblastoid cells in bone marrow.sup.6,7,8. Immune suppression caused by CAV infection is among the most important aspects of the disease.sup.9,10,11,12. In the field, CAV causes more serious problems when associated with other viruses.sup.13,14,15,16,17.
Current diagnostic tests for CAV are based on serological methods using neutralization tests.sup.6 , ELISA.sup.3 and immunofluorescence assays.sup.18. Noteborn et al..sup.19 reported two additional tests: polymerase chain reaction (PCR) and a dot-blot assay, using digoxigenin-labeled c-CAV DNA as probe on CAV isolates propagated in the lymphoblastoid T-cell line, MDCC-MSB1. Allan et al..sup.20 compared the immunocytochemical method for detection of CAV antibodies and in situ hybridization technique for localization of CAV on formalin-fixed and paraffin-embedded thymus tissue.
Prior to the present invention, isolation of CAV in cell cultures.sup.24 and the aforementioned immunofluorescence assays.sup.18, ELISA's.sup.3, and detection of virus by PCR and by in situ hybridization in formalin-fixed and paraffin-embedded thymus tissue.sup.20, have been methods of choice in laboratory diagnosis of chicken anemia. However, isolation of the virus is an extremely time-consuming procedure and not always the most appropriate because diagnosis is usually required in short period of time. Immunofluorescence assays can be carried out only if specialized equipment is available as well experienced personnel, which is often not the case in diagnostic laboratories. Serological tests should be supplemented by antigen or nucleic acid detection in cases of inadequate immune response to acute infection by immunocompromised individuals.
When immunocytochemical methods are used for detection of virus in tissue, the main obstacle is cross-linking of proteins and subsequent masking of viral antigen as a result of routine formalin fixation of tissue following post mortem examination. The loss of antigenicity can be somewhat reversed by treatment with proteolytic enzymes but this is of limited use for CAV.sup.20. In situ hybridization method for detection of CAV in tissue sections, although not impaired following long fixation time, requires conventional tissue preparation including paraffin embedment and tissue sectioning that delays diagnosis.
The present study describes in situ hybridization technique for detection of CAV DNA in blood smears. This method utilizes very simple and cost effective sample collection and preparation, and is applicable for wide use in the field for rapid diagnosis of any infectious agent that is present in whole blood cells.